Thioester-terminated water soluble polymers and method of modifying the N-terminus of a polypeptide therewith

ABSTRACT

The invention provides reagents and methods for conjugating a polymer specifically to the α-amine of a polypeptide. The invention provides monofunctional, bifunctional, and multifunctional PEGs and related polymers having a terminal thioester moiety capable of specifically conjugating to the α-amine of a polypeptide having a cysteine or histidine residue at the N-terminus. The invention provides reactive thioester-terminated PEG polymers that have suitable reactivity with an N-terminal cysteine or histidine residue of a polypeptide to produce an amide bond between the PEG molecule and the polypeptide.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/902,072, now U.S. Pat. No. 8,071,678, filed Oct. 11, 2010, which is acontinuation of U.S. patent application Ser. No. 12/372,727, now U.S.Pat. No. 7,834,088, filed Feb. 17, 2009, which is a continuation of U.S.patent application Ser. No. 11/353,656, filed Feb. 13, 2006, now U.S.Pat. No. 7,511,095, which is a divisional of U.S. patent applicationSer. No. 10/269,028, filed Oct. 9, 2002, now U.S. Pat. No. 7,078,496,which is a continuation-in-part of U.S. patent application Ser. No.09/973,318, filed Oct. 9, 2001, now U.S. Pat. No. 6,908,963, thedisclosures of which are incorporated herein by reference in theirentireties.

FIELD OF THE INVENTION

The invention relates to water soluble polymers useful for selectivelyconjugating to the N-terminus of a polypeptide.

BACKGROUND OF THE INVENTION

Covalent attachment of the hydrophilic polymer poly(ethylene glycol),abbreviated PEG, also known as poly(ethylene oxide), abbreviated PEO, tomolecules and surfaces is of considerable utility in biotechnology andmedicine. PEG is a polymer having the beneficial properties ofsolubility in water and in many organic solvents, lack of toxicity, andlack of immunogenicity. One use of PEG is to covalently attach thepolymer to water-insoluble molecules to improve the solubility of theresulting PEG-molecule conjugate. For example, it has been shown thatthe water-insoluble drug paclitaxel, when coupled to PEG, becomeswater-soluble. Greenwald, et al., J. Org. Chem., 60:331-336 (1995). PEGhas also been used increasingly in the modification of polypeptide andprotein therapeutics

The use of polypeptides, including proteins, for therapeuticapplications has expanded in recent years mainly due to both improvedmethods for recombinant expression of human polypeptides from variousexpression systems and improved methods of delivery in vivo. Many of thedrawbacks associated with polypeptide therapeutics, including shortcirculating half-life, immunogenicity and proteolytic degradation, havebeen improved by various approaches including gene therapy, epitopemutations by directed or shuffling mutagenesis, shielding of the epitoperegions by natural or synthetic polymers, fusion proteins, andincorporation of the polypeptide into drug delivery vehicles forprotection and slow release

Polymer modification of proteins, such as covalent attachment ofpoly(ethylene glycol), has gained popularity as a method to improve thepharmacological and biological properties of therapeutically usefulproteins. For example, certain poly(ethylene glycol) conjugated proteinshave been shown to have significantly enhanced plasma half-life, reducedantigenicity and immunogenicity, increased solubility and decreasedproteolytic degradation when compared to their non-pegylatedcounterparts. Factors that affect the foregoing properties are numerousand include the nature of the protein itself, the number ofpoly(ethylene glycol) or other polymer chains attached to the protein,the molecular weight and structure of the polymer chains attached to theprotein, the chemistries (i.e., the particular linkers) used to attachthe polymer to the protein, and the location of the polymermodified-sites on the protein.

To couple PEG to a molecule, such as a protein, it is often necessary to“activate” the PEG by preparing a derivative of the PEG having afunctional group at a terminus thereof. The functional group is chosenbased on the type of available reactive group on the molecule that willbe coupled to the PEG. For example, the functional group could be chosento react with an amino group on a protein in order to form a PEG-proteinconjugate

A variety of methods have been developed to non-specifically or randomlyattach poly(ethylene glycol) to proteins. Most commonly,electrophilically-activated poly(ethylene glycol) is reacted withnucleophilic side chains found of proteins. Attaching an activatedpoly(ethylene glycol) to the α-amine and ε-amine groups found on lysineresidues and at the N-terminus results in a mixture of conjugateproducts as described in U.S. Pat. No. 6,057,292. For example, theconjugate may consist of a population of conjugated proteins havingvarying numbers of poly(ethylene glycol) molecules attached to theprotein molecule (“PEGmers”), ranging from zero to the number of α- andε-amine groups in the protein. Often, random pegylation approaches areundesirable, due to variations in the ratios of PEG-mer productsproduced, and the desire, in certain cases, for a single, discretePEG-protein conjugate product. For a protein molecule that has beensingly modified by employing a non-site specific pegylation methodology,the poly(ethylene glycol) moiety may be attached at any one of a numberof different amine sites. Additionally, this type of non-specificPEGylation can result in partial or complete loss of the therapeuticutility of the conjugated protein, particularly for conjugates havingmore than one PEG attached to the protein.

Several methods for site-directed or selective attachment of PEG havebeen described. For example, WO 99/45026 suggests chemical modificationof a N-terminal serine residue to form an aldehyde functionalitysuitable for reaction with a polymer terminated with a hydrazide orsemicarbazide functionality. U.S. Pat. Nos. 5,824,784 and 5,985,265suggest reacting a polymer bearing a carbonyl group with the aminoterminus of a protein under reducing alkylation conditions and at a pHthat promotes selective attack at the N-terminus. WO 99/03887 and U.S.Pat. Nos. 5,206,344 and 5,766,897 relate to the site-directed PEGylationof cysteine residues that have been engineered into the amino acidsequence of proteins (cysteine-added variants). While these methodsoffer some advantages over non-specific attachment, there is acontinuing unmet need for improved methods and reagents for providingsite-specific polymer-conjugated proteins that do not require chemicalmodification of the polypeptide or careful control of certain reactionconditions, such as pH. Additionally, due to the high desirability formodifying a protein at its reactive amino-functionalities, there is aneed for improved polymer reagents that react selectively with aspecific protein amino group, such as the N-terminal amino group, forpreparing protein-polymer conjugates that are not a mixture ofPEG-polymer PEGmers but rather have PEG attached to a single, identifiedsite on the protein

SUMMARY OF THE INVENTION

This invention provides reagents and methods for conjugating polymersspecifically to the α-amino group of a polypeptide. The inventionprovides monofunctional, bifunctional, and multifunctional PEGs andrelated polymers having a thioester (also referred to as a thiol ester)moiety capable of specifically conjugating to the α-amine of apolypeptide having a cysteine or histidine at the N-terminus. Thus, theinvention provides reactive thioester-terminated PEG polymers effectiveto react site-specifically with an N-terminal cysteine or histidineresidue of a polypeptide to produce an amide-linked PEG-polypeptideconjugate

In one aspect, the invention provides a thioester-terminated reactivepolymer comprising a water soluble and non-peptidic polymer backbonehaving at least one terminus bonded to the structure:

wherein:

L is the point of bonding to a water soluble and non-peptidic polymerbackbone;

Z is a hydrolytically stable linkage or a hydrolytically unstablelinkage, such as O, S, —NHCO—, —CONH—, —O₂C—, —NHCO₂—, or —O₂CNH—;

a is 0 or 1;

each X is independently selected from H and alkyl, such as C1-C6 alkyl;

m is from 0 to about 12, preferably 1 to about 4;

Y is a heteroatom, preferably O or S; and

Q is a sulfur-containing leaving group preferably having the formula—S—R₁, wherein R₁ is hydrogen, alkyl, substituted alkyl, cycloalkyl,substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocycle, or substituted heterocycle.

The reactive polymer may be monofunctional (e.g., mPEG), bifunctional,or multifunctional. The polymer backbone is preferably a poly(alkyleneglycol), such as poly(ethylene glycol), poly(propylene glycol), or acopolymer of ethylene glycol and propylene glycol. Examples of othersuitable polymer backbones include poly(oxyethylated polyol),poly(olefinic alcohol), poly(vinylpyrrolidone), poly(α-hydroxy acid),poly(vinyl alcohol), polyphosphazene, polyoxazoline,poly(N-acryloylmorpholine), polyacrylate, polyacrylamides,polysaccharides, and copolymers, terpolymers, and mixtures thereof.

In another aspect, the invention provides a polymer conjugate of apolypeptide having a cysteine or histidine molecule at the N-terminus,the polymer conjugate comprising a water soluble and non-peptidicpolymer backbone having at least one terminus bonded to the structure:

wherein:

L, Z, m, Y, X and a are as defined above,

W is —CH₂SH or

and

POLYPEPTIDE is a polypeptide molecule, where —NH—C(W)H— represents theN-terminal cysteine or histidine residue (absent one hydrogen atom) ofthe polypeptide. Examples of polypeptides that can be conjugated to thethioester-terminated polymers of the invention include, but are notlimited to, protein ligands, enzymes, cytokines, hematopoietins, growthfactors, hormones, antigens, antibodies, antibody fragments, receptors,and protein fragments.

In yet another aspect, a method of conjugating a polymer derivative to apolypeptide having a cysteine or histidine molecule at the N-terminus isalso provided. The method comprises providing both a polypeptide havinga cysteine or histidine molecule at the N-terminus and athioester-terminated polymer as described above. The polypeptide isreacted with the thioester-terminated polymer to form, in a sitespecific manner, a conjugate having an amide linkage between the residueof the N-terminal histidine or cysteine molecule and the reactivepolymer. The thioester-terminated polymer selectively attaches to theN-terminal amine group of the histidine or cysteine residue of thepolypeptide without reacting with free amine groups at other positionswithin the polypeptide.

DETAILED DESCRIPTION OF THE INVENTION

The present invention now will be described more fully hereinafter. Thisinvention may, however, be embodied in many different forms and shouldnot be construed as limited to the embodiments set forth herein; rather,these embodiments are provided so that this disclosure will be thoroughand complete, and will fully convey the scope of the invention to thoseskilled in the art.

I. DEFINITIONS

The following terms as used herein have the meanings indicated.

As used in the specification, and in the appended claims, the singularforms “a”, “an”, “the”, include plural referents unless the contextclearly dictates otherwise.

The terms “functional group”, “active moiety”, “reactive site”,“chemically reactive group” and “chemically reactive moiety” are used inthe art and herein to refer to distinct, definable portions or units ofa molecule. The terms are somewhat synonymous in the chemical arts andare used herein to indicate the portions of molecules that perform somefunction or activity and are reactive with other molecules. The term“active,” when used in conjunction with functional groups, is intendedto include those functional groups that react readily with electrophilicor nucleophilic groups on other molecules, in contrast to those groupsthat require strong catalysts or highly impractical reaction conditionsin order to react (i.e., “non-reactive” or “inert” groups). For example,as would be understood in the art, the term “active ester” would includethose esters that react readily with nucleophilic groups such as amines.Exemplary active esters include N-hydroxysuccinimidyl esters or1-benzotriazolyl esters. Typically, an active ester will react with anamine in aqueous medium in a matter of minutes, whereas certain esters,such as methyl or ethyl esters, require a strong catalyst in order toreact with a nucleophilic group. As used herein, the term “functionalgroup” includes protected functional groups.

The term “protected functional group” or “protecting group” or“protective group” refers to the presence of a moiety (i.e., theprotecting group) that prevents or blocks reaction of a particularchemically reactive functional group in a molecule under certainreaction conditions. The protecting group will vary depending upon thetype of chemically reactive group being protected as well as thereaction conditions to be employed and the presence of additionalreactive or protecting groups in the molecule, if any. Protecting groupsknown in the art can be found in Greene, T. W., et al., P ROTECTIVEGROUPS IN ORGANIC SYNTHESIS, 3rd ed., John Wiley & Sons, New York, N.Y.(1999).

The term “linkage” or “linker” (L) is used herein to refer to an atom ora collection of atoms used to link, preferably by one or more covalentbonds, interconnecting moieties such two polymer segments or a terminusof a polymer and a reactive functional group present on a bioactiveagent, such as a polypeptide. A linker of the invention may behydrolytically stable or may include a physiologically hydrolyzable orenzymatically degradable linkage.

A “physiologically hydrolyzable” or “hydrolytically degradable” bond isa weak bond that reacts with water (i.e., is hydrolyzed) underphysiological conditions. Preferred are bonds that have a hydrolysishalf life at pH 8.25° C. of less than about 30 minutes. The tendency ofa bond to hydrolyze in water will depend not only on the general type oflinkage connecting two central atoms but also on the substituentsattached to these central atoms. Appropriate hydrolytically unstable ordegradable linkages include but are not limited to carboxylate ester,phosphate ester, anhydrides, acetals, ketals, acyloxyalkyl ether,imines, orthoesters, peptides and oligonucleotides.

A “hydrolytically stable” linkage or bond refers to a chemical bond,typically a covalent bond, that is substantially stable in water, thatis to say, does not undergo hydrolysis under physiological conditions toany appreciable extent over an extended period of time. Examples ofhydrolytically stable linkages include but are not limited to thefollowing: carbon-carbon bonds (e.g., in aliphatic chains), ethers,amides, urethanes, and the like. Generally, a hydrolytically stablelinkage is one that exhibits a rate of hydrolysis of less than about1-2% per day under physiological conditions. Hydrolysis rates ofrepresentative chemical bonds can be found in most standard chemistrytextbooks.

An “enzymatically unstable” or degradable linkage is a linkage that canbe degraded by one or more enzymes.

The term “polymer backbone” refers to the covalently bonded chain ofrepeating monomer units that form the polymer. For example, the polymerbackbone of PEG is—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—where n typically ranges from about 2 to about 4000. As would beunderstood, the polymer backbone may be covalently attached to terminalfunctional groups or pendant functionalized side chains spaced along thepolymer backbone.

The term “reactive polymer” refers to a polymer bearing at least onereactive functional group.

Unless otherwise noted, molecular weight is expressed herein as numberaverage molecular weight (M_(n)), which is defined as

$\frac{\sum{NiMi}}{\sum{Ni}},$wherein Ni is the number of polymer molecules (or the number of moles ofthose molecules) having molecular weight Mi.

The term “alkyl” refers to hydrocarbon chains typically ranging fromabout 1 to about 12 carbon atoms in length, preferably 1 to about 6atoms, and includes straight and branched chains. The hydrocarbon chainsmay be saturated or unsaturated.

“Cycloalkyl” refers to a saturated or unsaturated cyclic hydrocarbonchain, including bridged, fused, or spiro cyclic compounds, preferablycomprising 3 to about 12 carbon atoms, more preferably 3 to about 8.

The term “substituted alkyl” or “substituted cycloalkyl” refers to analkyl or cycloalkyl group substituted with one or more non-interferingsubstituents, such as, but not limited to, C3-C8 cycloalkyl, e.g.,cyclopropyl, cyclobutyl, and the like; acetylene; cyano; alkoxy, e.g.,methoxy, ethoxy, and the like; lower alkanoyloxy, e.g., acetoxy;hydroxy; carboxyl; amino; lower alkylamino, e.g., methylamino; ketone;halo, e.g. chloro or bromo; phenyl; substituted phenyl, and the like.

“Alkoxy” refers to an —O—R group, wherein R is alkyl or substitutedalkyl, preferably C1-C6 alkyl (e.g., methoxy or ethoxy).

“Aryl” means one or more aromatic rings, each of 5 or 6 core carbonatoms. Multiple aryl rings may be fused, as in naphthyl or unfused, asin biphenyl. Aryl rings may also be fused or unfused with one or morecyclic hydrocarbon, heteroaryl, or heterocyclic rings.

“Substituted aryl” is aryl having one or more non-interfering groups assubstituents. For substitutions on a phenyl ring, the substituents maybe in any orientation (i.e., ortho, meta or para).

“Heteroaryl” is an aryl group containing from one to four heteroatoms,preferably N, O, or S, or a combination thereof, which heteroaryl groupis optionally substituted at carbon or nitrogen atom(s) with C1-6 alkyl,—CF₃, phenyl, benzyl, or thienyl, or a carbon atom in the heteroarylgroup together with an oxygen atom form a carbonyl group, or whichheteroaryl group is optionally fused with a phenyl ring. Heteroarylrings may also be fused with one or more cyclic hydrocarbon,heterocyclic, aryl, or heteroaryl rings. Heteroaryl includes, but is notlimited to, 5-membered heteroaryls having one hetero atom (e.g.,thiophenes, pyrroles, furans); 5-membered heteroaryls having twoheteroatoms in 1, 2 or 1,3 positions (e.g., oxazoles, pyrazoles,imidazoles, thiazoles, purines); 5-membered heteroaryls having threeheteroatoms (e.g., triazoles, thiadiazoles); 5-membered heteroarylshaving 3 heteroatoms; 6-membered heteroaryls with one heteroatom (e.g.,pyridine, quinoline, isoquinoline, phenanthrine,5,6-cycloheptenopyridine); 6-membered heteroaryls with two heteroatoms(e.g., pyridazines, cinnolines, phthalazines, pyrazines, pyrimidines,quinazolines); 6-membered heteroaryls with three heteroatoms (e.g.,1,3,5-triazine); and 6-membered heteroaryls with four heteroatoms.

“Substituted heteroaryl” is heteroaryl having one or morenon-interfering groups as substituents.

“Heterocycle” or “heterocyclic” means one or more rings of 5-12 atoms,preferably 5-7 atoms, with or without unsaturation or aromatic characterand at least one ring atom which is not carbon. Preferred heteroatomsinclude sulfur, oxygen, and nitrogen. Multiple rings may be fused, as inquinoline or benzofuran.

“Substituted heterocycle” is heterocycle having one or more side chainsformed from non-interfering substituents.

“Non-interfering substituents are those groups that, when present in amolecule, are typically non-reactive with other functional groupscontained within the molecule.

Suitable non-interfering substituents or radicals include, but are notlimited to, halo, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C1-C10alkoxy, C7-C12 aralkyl, C7-C12 alkaryl, C3-C10 cycloalkyl, C3-C10cycloalkenyl, phenyl, substituted phenyl, toluoyl, xylenyl, biphenyl,C2-C12 alkoxyalkyl, C7-C12 alkoxyaryl, C7-C12 aryloxyalkyl, C6-C12oxyaryl, C1-C6 alkylsulfinyl, C1-C10 alkylsulfonyl, —(CH₂)_(m)—O—(C1-C10alkyl) wherein m is from 1 to 8, aryl, substituted aryl, substitutedalkoxy, fluoroalkyl, heterocyclic radical, substituted heterocyclicradical, nitroalkyl, —NO₂, —CN, —NRC(O)—(C1-C10 alkyl), —C(O)—(C1-C10alkyl), C2-C10 thioalkyl, —C(O)O—(C1-C10 alkyl), —OH, —SO₂, —S, —COOH,—NR, carbonyl, —C(O)—(C1-C10 alkyl)-CF₃, —C(O)—CF₃, —C(O)NR₂, —(C1-C10alkyl)-S—(C6-C12 aryl), —C(O)—(C6-C12 aryl),—(CH₂)_(m)—O—(CH₂)_(m)—O—(C1-C10 alkyl) wherein each m is from 1 to 8,—C(O)NR, —C(S)NR, —SO₂NR, —NRC(O)NR, —NRC(S)NR, salts thereof, and thelike. Each R as used herein is H, alkyl or substituted alkyl, aryl orsubstituted aryl, aralkyl, or alkaryl.

“Heteroatom” means any non-carbon atom in a hydrocarbon analog compound.Examples include oxygen, sulfur, nitrogen, phosphorus, arsenic, silicon,selenium, tellurium, tin, and boron.

The term “drug”, “biologically active molecule”, “biologically activemoiety” or “biologically active agent”, when used herein means anysubstance which can affect any physical or biochemical properties of abiological organism, including but not limited to viruses, bacteria,fungi, plants, animals, and humans. In particular, as used herein,biologically active molecules include any substance intended fordiagnosis, cure mitigation, treatment, or prevention of disease inhumans or other animals, or to otherwise enhance physical or mentalwell-being of humans or animals. Examples of biologically activemolecules include, but are not limited to, peptides, proteins, enzymes,small molecule drugs, dyes, lipids, nucleosides, oligonucleotides,polynucleotides, nucleic acids, cells, viruses, liposomes,microparticles and micelles. Classes of biologically active agents thatare suitable for use with the invention include, but are not limited to,antibiotics, fungicides, anti-viral agents, anti-inflammatory agents,anti-tumor agents, cardiovascular agents, anti-anxiety agents, hormones,growth factors, steroidal agents, and the like.

“Polyolefinic alcohol” refers to a polymer comprising a polyolefinbackbone, such as polyethylene, having multiple pendant hydroxyl groupsattached to the polymer backbone. An exemplary polyolefinic alcohol ispolyvinyl alcohol.

As used herein, “non-peptidic” refers to a polymer backbonesubstantially free of peptide linkages. However, the polymer backbonemay include a minor number of peptide linkages spaced along the lengthof the backbone, such as, for example, no more than about 1 peptidelinkage per about 50 monomer units.

“Polypeptide” or “poly(amino acid)” refers to any molecule comprising aseries of amino acid residues, typically at least about 10-20 residues,linked through amide linkages (also referred to as peptide linkages)along the alpha carbon backbone. While in some cases the terms may beused synonymously herein, a polypeptide is a peptide typically having amolecular weight up to about 10,000 Da, while peptides having amolecular weight above that are commonly referred to as proteins.Modifications of the peptide side chains may be present, along withglycosylations, hydroxylations, and the like. Additionally, othernon-peptidic molecules, including lipids and small drug molecules, maybe attached to the polypeptide. The polypeptide may comprise anycombination or sequence of amino acid residues. The polymers of theinvention are suitable for covalent attachment to both polypeptides andproteins.

“Amino acid” refers to organic acids containing both a basic amine groupand an acidic carboxyl group. The teem encompasses essential andnon-essential amino acids and both naturally occurring and synthetic ormodified amino acids. The most common amino acids are listed herein byeither their full name or by the three letter or single letterabbreviations: Glycine (Gly, G), Alanine (Ala, A), Valine (Val, V),Leucine (Leu, L), Isoleucine (Ile, I), Methionine (Met, M), Proline(Pro, P), Phenylalanine (Phe, F), Tryptophan (Trp, W), Serine (Ser, S),Threonine (Thr, T), Asparagine (Asn. N), Glutamine (Gln, Q), Tyrosine,(Tyr, Y), Cysteine (Cys, C), Lysine (Lys, K), Arginine (Arg, R),Histidine (His, H), Aspartic Acid (Asp, D), and Glutamic acid (Glu, E).

By “residue” is meant the portion of a molecule remaining after reactionwith one or more molecules. For example, an amino acid residue in apolypeptide chain is the portion of an amino acid remaining afterforming peptide linkages with adjacent amino acid residues.

“Oligomer” refers to short monomer chains comprising 2 to about 10monomer units, preferably 2 to about 5 monomer units.

The term “conjugate” is intended to refer to the entity formed as aresult of covalent attachment of a molecule, such as a biologicallyactive molecule, to a reactive polymer molecule, preferablypoly(ethylene glycol)

The term “leaving group” refers to an atom or collection of atomscovalently attached to an atom (such as a carbon atom) and that can bereadily displaced from the atom, taking with it its bonding electrons.Typically, the leaving group is an anion or a neutral molecule. Thebetter the leaving group, the more likely it is to depart from the atomto which it is bonded. Representative good leaving groups are those thatare the conjugate base of a strong acid.

“Multifunctional” in the context of a polymer of the invention means apolymer having 3 or more functional groups attached thereto, where thefunctional groups may be the same or different. Multifunctional polymersof the invention will typically comprise from about 3-100 functionalgroups, or from 3-50 functional groups, or from 3-25 functional groups,or from 3-15 functional groups, or from 3 to 10 functional groups, orwill contain 3, 4, 5, 6, 7, 8, 9 or 10 functional groups attached to thepolymer backbone.

II. THIOESTER POLYMERS

In one aspect, the present invention provides thioester-terminated watersoluble polymers capable of selectively reacting with the N-terminalamino group of a polypeptide to form a polymer-polypeptide conjugatecomprising a single water soluble polymer chain attached at theN-terminus. Such a polymer-polypeptide conjugate is referred to hereinas mono-substituted (meaning a polymer chain is substituted at only onesite of the polypeptide). Modification of a polypeptide at only a singlesite is beneficial because the likelihood of a significant reduction inbioactivity due to the presence of the polymer chain is lessened ascompared to indiscriminate or random polymer attachment at various andmultiple sites along the polypeptide chain. Moreover, the polymers andmethod provided herein for forming site-specific conjugates provide anadditional advantage over commonly employed prior art methods sincemultiple protection/deprotection steps to prevent reaction of thepolymer with other reactive groups/positions contained within thepolypeptide are unnecessary. Additionally, such site selectivemodification eliminates the need for additional conjugate purificationsteps to isolate particular (e.g., monopegylated) conjugate species.Thus, use of the thioester polymers of the invention can offer the aboveadvantages while additionally providing the beneficial properties ofwater-soluble polymer attachment, such as increased water solubility,enhanced plasma half-life, and decrease in proteolytic degradation ascompared to an unmodified polypeptide.

As explained in greater detail below, the thioester-terminated polymersof the invention selectively react with an N-terminal cysteine orhistidine residue of a polypeptide. Without being bound by theory, thereaction involves nucleophilic attack of the thioester group by eitherthe thiol side chain of a cysteine residue or the imidazole side chainof a histidine residue to form a thioester intermediate. The thioesterintermediate then undergoes a rapid rearrangement that results intransfer of the acyl group of the polymer to the terminal amine group ofthe polypeptide, thereby producing a peptide bond between the polymerand the N-terminus of the polypeptide. As would be understood, sinceonly an N-terminal cysteine or histidine residue would provide the sidechain necessary for the initial reaction step (e.g., attack on thepolymer thioester carbonyl carbon by a reactive thiol group of a proteinhaving an N-terminal cysteine), the polymers of the invention will, viaa molecular rearrangement, specifically attach to the N-terminal aminewithout reacting with any other side chain amine groups that may bepresent in the polypeptide molecule. The present invention isparticularly useful for site-specific PEG attachment of polypeptidescontaining more than one free cysteine or histidine, even in theunfolded state. The polymers and conjugation methods of the presentinvention can be used to assist insoluble polypeptides that are in theunfolded state to refold to their native conformation.

The thioester-terminated polymers of the invention comprise a polymerbackbone attached to a thioester group with an optional interveninglinkage between the terminus of the polymer backbone and the thioestergroup. The thioester-terminated polymers of the invention have thestructure:

wherein:

L is the point of bonding to a water soluble and non-peptidic polymerbackbone;

Z is a hydrolytically stable linkage or a hydrolytically unstablelinkage, such as O, S, —NHCO—, —CONH—, —O₂C—, —NHCO₂—, or —O₂CNH—;

m is from 0 to about 12, preferably 1 to about 4;

each X is independently selected from H and alkyl, such as C1-C6 alkyl;

a is 0 or 1;

Y is a heteroatom, preferably O or S; and

Q is a sulfur-containing leaving group preferably having the formula—S—R₁, wherein R₁ is hydrogen, alkyl, substituted alkyl, cycloalkyl,substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocycle, or substituted heterocycle.

A. Polymer Backbone

In general, the water soluble and non-peptidic polymer backbone shouldbe non-toxic and biocompatible, meaning that the polymer is capable ofcoexistence with living tissues or organisms without causing harm. Whenreferring to a thioester-terminated polymer backbone herein, it is to beunderstood that the polymer backbone can be any of a number of watersoluble and non-peptidic polymers, such as those described below.Preferably, poly(ethylene glycol) (PEG) is the polymer backbone. Theterm PEG includes poly(ethylene glycol) in any of a number of geometriesor forms, including linear forms (e.g., alkoxy PEG or bifunctional PEG),branched or multi-arm forms (e.g., forked PEG or PEG attached to apolyol core), pendant PEG, or PEG with degradable linkages therein, tobe more fully described below.

In its simplest form, PEG has the formula—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—  Formula IIwherein n is from about 10 to about 4000, typically from about 20 toabout 2000. Although the number average molecular weight of the PEGpolymer backbone can vary, PEGs having a number average molecular weightof from about 100 Da to about 100,000 Da, preferably about 5,000 Da toabout 60,000 Da are particularly useful. For example, PEG polymershaving a molecular weight of about 100 Da, about 200 Da, about 300 Da,about 500 Da, about 800 Da, about 1,000 Da, about 2,000 Da, about 3,000Da, about 4,000 Da, about 5,000 Da, about 10,000 Da, about 15,000, about20,000, about 30,000 and about 40,000 are useful in the presentinvention.

End-capped polymers, meaning polymers having at least one terminuscapped with a relatively inert group (e.g., an alkoxy group), can alsobe used as the polymer backbone of the invention. For example,methoxy-PEG-OH, or mPEG in brief, is a form of PEG wherein one terminusof the polymer backbone is bonded to a methoxy group, while the otherterminus is a hydroxyl group that is subject to ready chemicalmodification. The structure of mPEG is given below.CH₃O—(CH₂CH₂O)_(n)—CH₂CH₂—OH  Formula IIIwherein n is as described above.

Monomethoxy-terminated PEG molecules having a number average molecularweight of about 100 to about 100,000 Da, more preferably about 2,000 toabout 60,000 Da, are typically preferred for conjugating to proteins.Use of a monofunctional polymer such as mPEG prevents cross-linking ofthe protein that often occurs when bifunctional or multifunctionalreagents are used. In the present invention, mPEG-thioester can be usedto produce a single PEG molecule attached to a single protein molecule.However, in an alternate embodiment, utilizing a homobifunctionalPEG-thioester in appropriate proportions will result in a conjugatehaving two protein molecules attached to a single PEG molecule, even inthe event the protein contains multiple free cysteine residues. Due tothe manner in which the PEG derivative is believed to react (i.e.,initially linking through the available thiol group of the N-terminalcysteine residue and then rearranging to form the amide linkage), it isnot possible for the thioester polymer derivatives of the invention togive a cross-linked protein because other free cysteine residues willnot have both an available thiol group and an available amine group.Thus, another advantage of the present invention is the ability to usepolymers with multiple functional groups of the type described hereinwithout undesirable crosslinking with the polypeptide.

Multi-armed or branched PEG molecules, such as those described in U.S.Pat. No. 5,932,462, which is incorporated by reference herein in itsentirety, can also be used as the PEG polymer. For example, the PEGpolymer backbone can have the structure:

wherein:

poly_(a) and poly_(b) are PEG backbones, such as methoxy poly(ethyleneglycol);

R″ is a nonreactive moiety, such as H, methyl or a PEG backbone; and

P and Q are nonreactive linkages. In a preferred embodiment, thebranched PEG polymer is methoxy poly(ethylene glycol) disubstitutedlysine.

The PEG polymer may alternatively comprise a forked PEG. An example of aforked PEG is represented by PEG-YCHZ₂, where Y is a linking group and Zis an activated terminal group, such as the aldehyde group of thepresent invention, linked to CH by a chain of atoms of defined length.International Application No. PCT/US99/05333, the contents of which areincorporated by reference herein, discloses various forked PEGstructures capable of use in the present invention. The chain of atomslinking the Z functional groups to the branching carbon atom serve as atethering group and may comprise, for example, an alkyl chain, etherlinkage, ester linkage, amide linkage, or combinations thereof.

The PEG polymer may comprise a pendant PEG molecule having reactivegroups, such as carboxyl, covalently attached along the length of thePEG backbone rather than at the end of the PEG chain. The pendantreactive groups can be attached to the PEG backbone directly or througha linking moiety, such as an alkylene group.

In addition to the above-described forms of PEG, the polymer can also beprepared with one or more weak or degradable linkages in the polymerbackbone, including any of the above described polymers. For example,PEG can be prepared with ester linkages in the polymer backbone that aresubject to hydrolysis. As shown below, this hydrolysis results incleavage of the polymer into fragments of lower molecular weight:—PEG-CO₂—PEG-+H₂O→-PEG-CO₂H+HO-PEG-

Other hydrolytically degradable linkages, useful as a degradable linkagewithin a polymer backbone, include carbonate linkages; imine linkagesresulting, for example, from reaction of an amine and an aldehyde (see,e.g., Ouchi et al., Polymer Preprints, 38(1):582-3 (1997), which isincorporated herein by reference.); phosphate ester linkages formed, forexample, by reacting an alcohol with a phosphate group; hydrazonelinkages which are typically formed by reaction of a hydrazide and analdehyde; acetal linkages that are typically formed by reaction betweenan aldehyde and an alcohol; ortho ester linkages that are, for example,formed by reaction between a formate and an alcohol; peptide linkagesformed by an amine group, e.g., at an end of a polymer such as PEG, anda carboxyl group of a peptide; and oligonucleotide linkages formed by,for example, a phosphoramidite group, e.g., at the end of a polymer, anda 5′ hydroxyl group of an oligonucleotide.

It is understood by those skilled in the art that the term poly(ethyleneglycol) or PEG represents or includes all the above forms of PEG.

Many other polymers are also suitable for the invention. Any of avariety of monofunctional, bifunctional or multifunctional polymerbackbones that are non-peptidic and water-soluble could be used in thepresent invention. The polymer backbone can be linear, or may be in anyof the above-described forms (e.g., branched, forked, and the like).Examples of suitable polymers include, but are not limited to, otherpoly(alkylene glycols), copolymers of ethylene glycol and propyleneglycol, poly(olefinic alcohol), poly(vinylpyrrolidone),poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate),poly(saccharides), poly(α-hydroxy acid), poly(vinyl alcohol),polyphosphazene, polyoxazoline, poly(N-acryloylmorpholine), such asdescribed in U.S. Pat. No. 5,629,384, which is incorporated by referenceherein in its entirety, and copolymers, terpolymers, and mixturesthereof.

B. Linkage Between Polymer Backbone and Thioester

The intervening linkage between the terminus of the polymer backbone andthe thioester group is the residue of the functional group on thepolymer backbone that couples the polymer backbone to the terminalthioester group. Thus, as would be understood, the structure of thelinkage will vary depending on the structure of the functional group ofthe polymer backbone. The linkage can comprise a hydrolytically stablelinkage, such as amide, urethane, ether, thioether, or urea.Alternatively, the linkage can comprise a hydrolytically unstablelinkage, such as carboxylate ester, phosphate ester, orthoester,anhydride, imine, acetal, ketal, oligonucleotide, or peptide. In oneembodiment, in addition to the hydrolytically stable or unstablelinkage, the linkage between the polymer backbone and the thioesterincludes an optional alkylene spacer, designated herein as (X—CH)_(m).

As shown above in Formula I, the linkage preferably has the structure:

wherein:

Z is the hydrolytically stable or unstable linkage, such as

O, S, —NHCO—, —CONH—, —O₂C—, —NHCO₂—, or —O₂CNH—;

m is from 0 to about 12, preferably 1 to about 4;

each X is independently selected from H and alkyl, such as C1-C6 alkyl;and

a is 0 or 1.

The length of the alkylene chain (i.e., the value of m) can vary from 0to about 12. For example, m can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,or 12. Preferably, m is 0, 1, 2, 3, or 4. Each X of the alkylene chainis preferably hydrogen, methyl or ethyl. In a preferred embodiment, a isI and Z is a heteroatom, such as O or S.

C. Thioester Functional Group

The thioester functional group is covalently attached to at least oneterminus of the water soluble polymer. The thioester group has thestructure:

wherein:

Y is a heteroatom, preferably O or S; and

Q is a sulfur-containing electrophilic leaving group preferably havingthe formula —S—R₁,

wherein R₁ is hydrogen, alkyl, substituted alkyl, cycloalkyl,substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocycle, or substituted heterocycle.

The particular R₁ group employed can vary. The R₁ group, in conjunctionwith the sulfur atom, forms an electrophilic leaving group suitable fordisplacement during nucleophilic attack of the carbonyl carbon by thethiol or imidazole side chain of the N-terminal amino acid residue of apolypeptide. Preferred R₁ groups include substituents derived fromphenol, nitrophenol, benzoic acid, pyridine, pyridinecarboxylic acid,and nitropyridine. Substituted or unsubstituted pyridinyl isparticularly preferred. Examples 1-3 illustrate thioester-terminated PEGpolymers bearing a thiolpyridinyl leaving group.

D. Exemplary Polymer Structures

An embodiment of a linear polymer of the invention can be structurallyrepresented as shown below:

wherein POLY is a water soluble and non-peptidic polymer backbone, R isa capping group or a functional group, and Z, X, Y, m, a and Q are asdefined above. In a preferred embodiment, R is methoxy, POLY ispoly(ethylene glycol), a is 1, Z is O, m is 1 to about 3, Y is O, andeach X is H or CH₃.

The R group can be a relatively inert capping group, such as alkoxy(e.g., methoxy or ethoxy), alkyl, benzyl, aryl, or aryloxy (e.g.,benzyloxy). Alternatively, the R group can be a functional group capableof readily reacting with a functional group on a biologically activemolecule. Exemplary functional groups include hydroxyl, active ester(e.g. N-hydroxysuccinimidyl ester or 1-benzotriazolyl ester), activecarbonate (e.g. N-hydroxysuccinimidyl carbonate and 1-benzotriazolylcarbonate), acetal, aldehyde, aldehyde hydrate, alkenyl, acrylate,methacrylate, acrylamide, active sulfone, amine, hydrazide, thiol,carboxylic acid, isocyanate, isothiocyanate, maleimide, vinylsulfone,dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxal, dione,mesylate, tosylate, or tresylate. Specific examples of terminalfunctional groups for the polymer backbones of the invention includeN-succinimidyl carbonate (see e.g., U.S. Pat. Nos. 5,281,698,5,468,478), amine (see, e.g., Buckmann et al. Makromol. Chem. 182:1379(1981), Zalipsky et al. Eur. Polym. J. 19:1177 (1983)), hydrazide (See,e.g., Andresz et al. Makromol. Chem. 179:301 (1978)), succinimidylpropionate and succinimidyl butanoate (see, e.g., Olson et al. inPoly(ethylene glycol) Chemistry & Biological Applications, pp 170-181,Harris & Zalipsky Eds., ACS, Washington, D.C., 1997; see also U.S. Pat.No. 5,672,662), succinimidyl succinate (See, e.g., Abuchowski et al.Cancer Biochem. Biophys. 7:175 (1984) and Joppich et al., Makromol.Chem. 180:1381 (1979), succinimidyl ester (see, e.g., U.S. Pat. No.4,670,417), benzotriazole carbonate (see, e.g., U.S. Pat. No.5,650,234), glycidyl ether (see, e.g., Pitha et al. Eur. J. Biochem.94:11 (1979), Elling et al., Biotech. Appl. Biochem. 13:354 (1991),oxycarbonylimidazole (see, e.g., Beauchamp, et al., Anal. Biochem.131:25 (1983), Tondelli et al. J. Controlled Release 1:251 (1985)),p-nitrophenyl carbonate (see, e.g., Veronese, et al., Appl. Biochem.Biotech., 11:141 (1985); and Sartore et al., Appl. Biochem. Biotech.,27:45 (1991)), aldehyde (see, e.g., Harris et al. J. Polym. Sci. Chem.Ed. 22:341 (1984), U.S. Pat. No. 5,824,784, U.S. Pat. No. 5,252,714),maleimide (see, e.g., Goodson et al. Bio/Technology 8:343 (1990), Romaniet al. in Chemistry of Peptides and Proteins 2:29 (1984)), and Kogan,Synthetic Comm. 22:2417 (1992)), orthopyridyl-disulfide (see, e.g.,Woghiren, et al. Bioconj. Chem. 4:314 (1993)), acrylol (see, e.g.,Sawhney et al., Macromolecules, 26:581 (1993)), vinylsulfone (see, e.g.,U.S. Pat. No. 5,900,461). All of the above references are incorporatedherein by reference.

In a homobifunctional embodiment of Formula V, R is athioester-containing moiety of formula —(Z)_(a)—(CXH)_(m)—CO—S—R₁,wherein Z, a, x, m, and R₁ are as defined above.

Some specific examples of linear polymers of the invention are shownbelow:

wherein R₁ and n are as defined above.

One example of a multi-arm embodiment of the thioester-terminatedpolymer of the invention has the structure:

wherein each POLY is a water soluble and non-peptidic polymer backbone,R′ is a central core molecule, y is from about 3 to about 100,preferably 3 to about 25, and Z, X, Y, m, a and R₁ are as defined above.The core moiety, R′, is a residue of a molecule selected from the groupconsisting of polyols, polyamines, and molecules having a combination ofalcohol and amine groups. Specific examples of central core moleculesinclude glycerol, glycerol oligomers, pentaerythritol, sorbitol, andlysine.

The central core molecule is preferably a residue of a polyol having atleast three hydroxyl groups available for polymer attachment. A “polyol”is a molecule comprising a plurality of available hydroxyl groups.Depending on the desired number of polymer aims, the polyol willtypically comprise 3 to about 25 hydroxyl groups. The polyol may includeother protected or unprotected functional groups as well withoutdeparting from the invention. Although the spacing between hydroxylgroups will vary from polyol to polyol, there are typically 1 to about20 atoms, such as carbon atoms, between each hydroxyl group, preferably1 to about 5. Preferred polyols include glycerol, reducing sugars suchas sorbitol, pentaerythritol, and glycerol oligomers, such ashexaglycerol. A 21-arm polymer can be synthesized usinghydroxypropyl-β-cyclodextrin, which has 21 available hydroxyl groups.The particular polyol chosen will depend on the desired number ofhydroxyl groups needed for attachment to the polymer arms.

E. Method of Forming Thioester Polymers

The thioester polymers of the invention may be formed by derivatizationof a water-soluble non-peptidic polymer by any of a number of syntheticapproaches for forming thioesters known in the art. See, for example,Field, L. Synthesis, 1972, 106. For instance, a thioester can beprepared from the corresponding acid chloride-terminated polymer byreaction with a thallium(I) salt of a thiolate (Spessard, G., et al.,Organic Synthesis Collection, Vol. 7, 87). For thioester derivatizationof a polymer having additional functional groups contained within themolecule, such as hydroxy or other functional groups, alternativeapproaches such as the following may be employed. For example, athioester-terminated polymer as described herein can be formed from thecorresponding carboxylic acid-terminated polymer by reaction of the acidwith a dialkyl or diphenyl phosphorochloridate to form the anhydride,which can then be converted to the corresponding thioester. (Masamune,S., et al., Can. J. Chem., 1975, 53, 3693; Yamada, S., et al., Chem.Pharm. Bull. 1977, 25, 2423). In yet another synthetic approach, athioester-terminated polymer can be prepared by reaction of animidazolide of a carboxylic acid (prepared by reaction of thecorresponding carboxylic acid with N,N-carbonyldiimidazole) with arelatively acidic thiol (Masamune, S., et al., J. Am. Chem. Soc., 1976,98, 7874). Alternatively, a disulfide and triphenylphosphine can be usedto convert a carboxylic acid terminus of a polymer to the correspondingthioester (Mukaiyama, T., et al., Bull. Chem. Soc. Jpn., 1970, 43,1271). Other methods that can be used to prepare thioesters fromcarboxylic acids include the use of aryl thiocyanates (Grieco, P., etal., J. Org. Chem., 1978, 43, 1283), thiopyridyl chloroformate (Corey,E. J., et al., Tetrahedron Lett., 1979, 2875),2-fluoro-N-methylpyridinium tosylate (Watanabe, Y., et al., Chem. Lett.1976, 741), 1-hydroxybenzotrizaole (Horiki, K., Synth. Commun. 1977, 7,251), and boron thiolate (Pelter, A., et al., J. Chem. Soc., PerkinTrans. I, 1977, 1672). Alternatively, a polymer having an O-esterterminus can be converted to the corresponding S-ester by aluminum andboron reagents.

A preferred method of forming the thioester polymers of the inventioninvolves base-catalyzed reaction of a terminal carboxylic acid, oractive ester thereof, of a reactive polymer with a thiol compound offormula R₁—SH, wherein R₁ is as defined above. Preferred reactivepolymers bearing a terminal carboxylic acid group include poly(ethyleneglycol) terminated with a carboxymethyl, propionic acid, or butanoicacid group. Any other method known in the art for coupling a thioestergroup to a terminus of a polymer backbone, such as any of thosedescribed above, could also be used without departing from the presentinvention. Exemplary methods of forming thioester-terminated polymersare illustrated in Examples 1-3.

III. POLYMER/POLYPEPTIDE CONJUGATES

A. Structure of Polymer/Polypeptide Conjugate

The thioester polymers of the invention selectively react with theα-amine of a polypeptide having a histidine or cysteine molecule at theN-terminus to form an amide linkage between the polymer and thepolypeptide. In a preferred embodiment, the polymer-polypeptideconjugate comprises a water soluble and non-peptidic polymer backbonehaving at least one terminus bonded to the structure:

wherein:

L, Z, Y, m, X and a are defined above;

W is —CH₂SH or

depending on whether the terminal amino acid is cysteine or histidine;and

POLYPEPTIDE is the residue of the polypeptide molecule. The polymerbackbone can comprise any of the polymer structures discussed above,such as PEG in any of its forms.

The polypeptide can be any polypeptide having an N-terminal cysteine orhistidine residue, regardless of whether the N-terminal cysteine orhistidine is naturally occurring in the polypeptide or introduced bymodification of the polypeptide sequence. The polypeptide molecule ispreferably selected from the group consisting of proteins,protein-ligands, enzymes, cytokines, hematopoietins, growth factors,hormones, antigens, antibodies, antibody fragments, receptors, andprotein fragments. The following is an illustrative although by no meansexhaustive list of polypeptide molecules that include, or could bemodified to include, an N-terminal cysteine or histidine residue:calcitonin, parathyroid hormone, interferon alpha, interferon beta,interferon gamma, interleukins 1-21, granulocyte-colony stimulatingfactor, macrophage-colony stimulating factor, granulocyte-macrophagecolony stimulating factor, stem cell factor, leukemia inhibitory factor,kit-ligand, flt-3 ligand, erythropoietin, thrombopoietin, tumor necrosisfactor alpha, tumor necrosis factor beta, transforming growth factor,bone morphogenic proteins, osteoprotegerin, tissue plasminogenactivator, platelet derived growth factor, fibroblast growth factor,keratinocyte growth factor, epidermal growth factor, human growthhormone, insulin, tumor necrosis factor-related apoptosis-inducingligand (TRAIL), DNAse, receptors, enzymes, fusion proteins, chimericantibodies, humanized antibodies, fully human antibodies, Fab fragments,F(ab′)₂ fragments, Fv fragments, and scFv fragments. In one preferredembodiment, the polypeptide is an interferon molecule.

An exemplary embodiment of a linear polymer conjugate of the inventionhas the structure:

wherein R, POLY, Z, a, X, m, Y and W are as defined above.

In an alternative embodiment where the polymer is a multi-arm polymer,an exemplary polymer conjugate of the invention has the structure:

wherein R′, y, POLY, Z, a, X, m, Y and W are as defined above.

Polypeptide conjugates in accordance with the invention will possess anamide linkage formed by reaction with an N-terminal cysteine orhistidine of the polypeptide, where the polymer portion of the conjugatemay have any of a number of different geometries (e.g., linear,branched, forked, and the like), molecular weights, optional degradablelinkages, etc., as described in detail herein and in the accompanyingexamples. Representative conjugates prepared in accordance with theinvention are provided in Examples 4-7.

B. Method of Forming Polymer/Polypeptide Conjugate

The present invention uses a thioester-terminated polymer, such as athioester-terminated PEG, to specifically modify the α-amine of anN-terminal cysteine or histidine without permanently modifying theremaining free functional group (e.g., the thiol group of a cysteineresidue) on the terminal amino acid or modifying other amine groupspresent in the polypeptide chain. Although not bound by any particulartheory, Reaction Scheme I below illustrates the reaction believed tooccur between a polypeptide having an N-terminal cysteine molecule and areactive polymer of the invention. As shown, it is believed that thethioester-terminated polymer initially reacts with the free thiol groupof the cysteine and thereafter undergoes an intramolecular rearrangementto form an amide linkage with the N-terminal amine group, thus leavingthe thiol group available for further modification if desired. Thethiol-thioester exchange is preferably effected by use of atrialkylphosphine, such as tris(2-carboxyethyl)phosphine ortriethylphosphine, and optionally a thiol, such as mercaptopropionicacid.

Optionally, in the case of an N-terminal cysteine molecule, a secondthiol-reactive polymer (e.g., a thiol-reactive PEG) may be reacted withthe free thiol group in order to form a branched structure at theN-terminus of the polypeptide as shown in Reaction Scheme I, wherein L′is the linker resulting from the reaction of the thiol-reactive terminalfunctional group of the second PEG polymer with the free thiol group onthe cysteine molecule. In one embodiment, only two polymer backbones areattached to the polypeptide.

Examples of thiol-reactive functional groups include vinylsulfone,maleimide, orthopyridyl disulfide and iodoacetamide. Examples of the L′linkage include:

As would be readily understood by one of ordinary skill in the art, themethod of the invention could be used to couple the above-describedpolymer derivatives to any moiety, whether peptidic or not, having aterminal —CH(W)—NH₂ group, wherein W is as defined above.

IV. EXAMPLES

The following examples are given to illustrate the invention, but shouldnot be considered in limitation of the invention. For example, althoughmPEG is used in the examples to illustrate the invention, other forms ofPEG and similar polymers that are useful in the practice of theinvention are encompassed by the invention as discussed above.

All PEG reagents referred to in the appended examples are available fromShearwater Corporation of Huntsville, Ala. All ¹HNMR data was generatedby a 300 or 400 MHz NMR spectrometer manufactured by Bruker.

Examples 1-3 illustrate methods of forming a thioester-terminatedpolymer of the invention. Examples 4-7 illustrate reaction of athioester-terminated polymer of the invention with an exemplarypolypeptide having a N-terminal cysteine residue. As indicated below,use of the thioester polymers of the invention results in selectiveattachment of the polymer to the N-terminal amine of the polypeptide.

Example 1 Preparation of PEG (5000)-α-methoxy-co-propionic acid,2-pyridylthioester (PEG-PA-OPTE)

2-mercaptopyridine (40.0 mg, 0.36 mmoles), 1-hydroxybenzotriazole (4.0mg, 0.030 mmoles), 4-(dimethylamino)pyridine (36.7 mg, 0.30 mmoles) and1,3-dicyclohexylcarbodiimide (dissolved in 2 mL anhydrousdichloromethane, 84.0 mg, 0.41 mmoles) were added to a solution of PEG(5000)-α-methoxy-ω-propionic acid (1.5 g, 0.27 mmoles) in anhydrousacetonitrile (20 mL). The reaction solution was stirred overnight atambient temperature under argon. The solution was then concentrated tonear dryness at reduced pressure, followed by addition of anhydroustoluene (50 mL). The mixture was stirred at room temperature for thirtyminutes, filtered and the filtrate was concentrated at reduced pressureto near dryness. Ethyl acetate (200 mL) was added and the mixture waswarmed until the contents were completely dissolved. The solution wasthen cooled to room temperature while stirring. Ethyl ether (50 mL) wasadded and a precipitate formed. The product was filtered and rinsed withethyl ether until the product became white. The product was then driedunder high vacuum. Yield: 1.1 g. NMR (d6-DMSO): δ2.98 ppm (t, 2H,—CH₂—COS—), δ3.51 ppm (s, PEG backbone), δ7.46 ppm (m, ill resolved, 1H,H₅ (pyridyl)), δ7.64 ppm (d, 1H, H₃ (pyridyl)), δ7.91 ppm (t, 1H, H₄(pyridyl)), δ8.60 ppm (d, 1H, H₆ (pyridyl)).

Example 2 Preparation of PEG (5000)-α-benzyloxy(BZO)-ω-carboxymethyl,2-pyridylthioester (PEG-CM-OPTE)

2-mercaptopyridine (40.0 mg, 0.36 mmoles), 1-hydroxybenzotriazole (5.0mg, 0.035 mmoles), and 1,3-dicyclohexylcarbodiimide (dissolved in 2 mLanhydrous dichloromethane, 74.3 mg, 0.36 mmoles) were added to asolution of PEG (5000)-α-benzyloxy-ω-carboxymethyl (1.5 g, 0.30 mmoles)in anhydrous acetonitrile (20 mL). The reaction solution was stirredovernight at ambient temperature under argon. The solution was thenconcentrated to near dryness at reduced pressure, followed by additionof anhydrous toluene (30 mL). The mixture was stirred at roomtemperature for thirty minutes, filtered and the filtrate wasconcentrated at reduced pressure to near dryness. Ethyl acetate (150 mL)was added and the mixture was warmed until the contents were completelydissolved. The solution was then cooled to room temperature whilestirring. Ethyl ether (50 mL) was added to the solution and aprecipitate formed. The product was filtered and rinsed with ethyl etheruntil the product became white. The product was then dried under highvacuum. Yield: 1.1 g. NMR (d6-DMSO): δ3.51 ppm (s, PEG backbone), δ4.39ppm (s, 2H, —OCH₂COS—), δ4.49 ppm (s, 2H, —OCH₂-(benzyloxy)), δ7.33 ppm(m, ill resolved, 5H, C₆H₅ (benzyloxy)), δ7.46 ppm (m, ill resolved, 1H,H₅ (pyridyl)), δ7.63 ppm (d, 1H, H₃ (pyridyl)), δ7.91 ppm (t, 1H, H₄(pyridyl)), δ8.60 ppm (d, 1H, H₆ (pyridyl)).

Example 3 Preparation of PEG (5000)-α-methoxy-ω-2-methyl butanoic acid,2-pyridylthioester

2-mercaptopyridine (44.5 mg, 0.40 mmoles), 1-hydroxybenzotriazole (4.7mg, 0.033 mmoles), 4-(dimethylamino)pyridine (40.7 mg, 0.33 mmoles) and1,3-dicyclohexylcarbodiimide (dissolved in 2 mL anhydrousdichloromethane, 92.8 mg, 0.45 mmoles) were added to a solution of PEG(5000)-α-methoxy-ω-2-methyl butanoic acid (1.5 g, 0.30 mmoles) inanhydrous acetonitrile (20 mL). The reaction solution was stirredovernight at ambient temperature under argon. The solution was thenconcentrated to near dryness at reduced pressure, followed by additionof anhydrous toluene (50 mL). The mixture was stirred at roomtemperature for thirty minutes, filtered and the filtrate wasconcentrated at reduced pressure to near dryness. Ethyl acetate (150 mL)was added and mixture was warmed until the contents completelydissolved. The solution was then cooled to room temperature whilestirring. A precipitate was formed by adding 2-Propanol (50 mL),followed by addition of ethyl ether (50 mL). The product was filteredoff, rinsed with 2-propanol until the product became white. The productwas then dried under high vacuum. Yield: 1.2 g. NMR (d6-DMSO): δ1.19 ppm(d, 3H, —O—CH₂—CH₂—CH(CH₃)—COS—), δ1.66 ppm and δ1.92 ppm (m, 2H,—O—CH₂—CH₂—CH(CH₃)—COS—), δ2.89 ppm (m, 1H, —O—CH₂—CH₂—CH(CH₃)—COS—),δ3.51 ppm (s, PEG backbone), δ7.46 ppm (m, ill resolved, 1H, H₅(pyridyl)), δ7.63 ppm (d, 1H, H₃ (pyridyl)), δ7.90 ppm (t, 1H, H₄(pyridyl)), δ8.60 ppm (d, 1H, H₆ (pyridyl)).

Example 4 Conjugation of PEG-CM-OPTE to Interferon

Interferon tau (0.45 mg), which has a cysteine as the N-terminal aminoacid, was formulated to 0.3 mg/ml in 1M Tris, 0.7 mM TCEP(Tris[2-carboxyethylphosphine]hydrochloride) and 3 mM mercaptopropionicacid at pH 7.75. Approximately 1.0 mg of mPEG_(5K)-CM-OPTE (from Example2) was added to the interferon solution and allowed to react at roomtemperature for 4 hours. The reaction mixture was dialyzed againstdeionized water overnight. The product was analyzed by MALDI-MS. Themass spectrum showed free PEG at 5000 Da, unconjugated interferon at19,979 Da and a single PEG conjugate at a molecular weight of 25,065 Da,meaning the PEGylated product has only a single PEG molecule attached tothe polypeptide at the N-terminus.

Example 5 Conjugation of PEG-PA-OPTE to Interferon

Interferon tau (0.45 mg) was formulated to 0.3 mg/ml in 0.33M Tris, 0.7mM TCEP (Tris[2-carboxyethylphosphine]hydrochloride) at pH 7.75.Approximately 1.0 mg of mPEG_(SK)-PA-OPTE (orthopyridyl thioester ofpropionic acid from Example 1) was added to the interferon solution andallowed to react at room temperature for 4 hours. The product wasanalyzed by SDS-PAGE. The gel showed two bands corresponding tounconjugated interferon (˜20 kDa) and singly PEG-conjugated interferon(˜29 kDa) (i.e., a polypeptide attached to a single PEG molecule). Theslower migration of the PEG-interferon conjugate is due to the largerhydrodynamic volume of the PEG chain when compared to a correspondingmolecular weight protein.

Example 6 Conjugation of PEG-CM-OPTE to a Polypeptide

The polypeptide CRASKSVSSSGYSYMHWYQQ (MW=2355 Da) was formulated to 0.67mg/ml in 0.67M Tris, 1.3 mM TCEP(Tris[2-carboxyethylphosphine]hydrochloride) and 5.3M urea at pH 7.75.Approximately 21.0 mg of mPEG_(5K)-CM-OPTE (from Example 2) was added tothe polypeptide solution and allowed to react at room temperature for 4hours. The reaction mixture was dialyzed against deionized waterovernight. The product was analyzed by MALDI-MS. The mass spectrumshowed a conjugate comprising a single PEG molecule attached to thepolypeptide and having a molecular weight of 7555 Da. This demonstratesthat the thioester-terminated polymer did not randomly react with otherfree amine groups in the molecule, such as the amine groups of thelysine or arginine residues.

Example 7 Conjugation of PEG-PA-OPTE to a Polypeptide

The polypeptide CRASKSVSSSGYSYMHWYQQ (MW=2355 Da) was formulated to 0.67mg/ml in 0.67M Tris, 1.3 mM TCEP(Tris[2-carboxyethylphosphine]hydrochloride) and 5.3M urea at pH 7.75.Approximately 21.0 mg of mPEG_(5K)-PA-OPTE (from Example 1) was added tothe polypeptide solution and allowed to react at room temperature for 4hours.

Many modifications and other embodiments of the invention will come tomind to one skilled in the art to which this invention pertains havingthe benefit of the teachings presented in the foregoing description.Therefore, it is to be understood that the invention is not to belimited to the specific embodiments disclosed and that modifications andother embodiments are intended to be included within the scope of theappended claims. Although specific terms are employed herein, they areused in a generic and descriptive sense only and not for purposes oflimitation.

1. A composition comprising a conjugate having the structure:

wherein: R′ is a central core molecule; POLY is a water-soluble andnon-peptidic polymer backbone; Z is a linker; a is 0 or 1; m is from 0to about 12; each X is independently selected from H and alkyl; Y is aheteroatom; W is CH₂SH or

POLYPEPTIDE is a residue of a polypeptide molecule; and y is from 3 toabout
 100. 2. The composition of claim 1, wherein the central coremolecule, R′, is a residue of a polyol having at least three hydroxylgroups.
 3. The composition of claim 2, wherein the polyol is selectedfrom the group consisting of glycerol, hexaglycerol, sorbitol andpentaerythritol.
 4. The composition of claim 3, wherein the polyol ispentaerythritol.
 5. The composition of claim 1, wherein thewater-soluble and non-peptidic polymer backbone, POLY, is apoly(ethylene glycol).
 6. The composition of claim 1, wherein thepoly(ethylene glycol) has a molecular weight of about 100 Da to about100,000 Da.
 7. The composition of claim 1, wherein the linker, Z, isselected from the group consisting of —O—, —S—, —NHCO—, —CONH—, —O₂C—,—NH—CO₂— and —O₂CNH—.
 8. The composition of claim 1, wherein the linker,Z, is —NHCO—.
 9. The composition of claim 1, wherein m is 1 to about 4.10. The composition of claim 8, wherein m is
 2. 11. The composition ofclaim 1, wherein the hetereoatom, Y, is O or S.
 12. The composition ofclaim 11, wherein the heteroatom, Y, is O.
 13. The composition of claim1, wherein W is CH₂SH.
 14. The composition of claim 1, wherein W is


15. The composition of claim 1, wherein y is 3 to about
 25. 16. Thecomposition of claim 15, wherein y is 3-10.
 17. The composition of claim16, wherein y is 4.